OBJECTIVES: Pentavalent antimonials have been used for the treatment of leishmaniasis for over 70 years, however they are limited by their toxicity. Unfortunately, the efficacy of first-line drugs for the treatment of leishmaniasis has decreased and resistance is noticeable. Luliconazole is a new azole with unique effects on fungi that has not yet been tested on Leishmania parasites. METHODS: In this study, the cytotoxicity and antileishmanial activity of luliconazole were evaluated in vitro against promastigotes and intracellular amastigotes of Leishmania major. The docking simulation with the target enzyme, sterol 14α-demethylase (CYP51) was performed using AutoDock 4.2 program. RESULTS: The IC (concentration of test compound required for 50% inhibition) against promastigotes revealed that luliconazole (IC=0.19μM) has greater potency than ketoconazole (KET), meglumine antimoniate (MA) and amphotericin B (AmB) (IC values of 135, 538 and 2.52μM, respectively). Against the amastigote stage, luliconazole at a concentration of 0.07μM decreased the mean infection rate and the mean number of amastigotes per macrophage more effectively than MA (P<0.004) and KET (P<0.043), but there was no difference compared with AmB (P>0.05). A docking study of luliconazole with the cytochrome P450 enzyme sterol 14α-demethylase (CYP51) revealed that this azole drug can properly interact with the target enzyme in Leishmania mainly via coordination with heme and multiple hydrophobic interactions. CONCLUSION: These results show the potent activity of luliconazole at extremely low concentrations against L. major. It may therefore be considered as a new candidate for treatment of leishmaniasis in the near future.